Abstract: Objective To establish a system for the specific knockout genes of testis tissue in vivo. Methods The sgRNA functional elements in the CRISPR/Cas9 system were inserted into the AAV expression vector by homologous recombination technology, and the Wee1 2# sgRNA was constructed into the modified AAV-sgRNA (new version) vector for virus packaging. The efficiency of gene knockout of this vector was verified by infection with Hela-spCas9 cells stably expressing Cas9. The sgRNA target of the testis tissue-specific gene Sycp3 was screened and constructed into AAV-sgRNA (new version) vector for viral packaging. The virus was injected into the seminiferous tubule of mouse testicular tissue by microinjection technique, and the in vivo cells were analyzed by T7E1. Gene knockout effect. Results The successful construction of the AAV-sgRNA vector was able to perform viral packaging and gene editing of the human Wee1 gene at the cellular level in vitro, which was not significantly different from the vector editing efficiency in the Lentivirus-mediated CRISPR/Cas9 system. At the same time, the system can genetically edit Sycp3 at the level of the body. Conclusions A system for specifically knocking out target genes in testis tissue in vivo has been successfully established, which provides a new idea and method for the study of reproductive function in vivo.

Key words: CRISPR Cas9, AAV, virus packaging, T7E1，Microinjection